1. Field of the Invention
The present invention relates to reversed and normal stationary phases for liquid chromatography and methods for their production.
2. Description of the Prior Art
Reversed and normal phase bonded metal oxides, particularly silica, are the most popular packings used in high performance liquid chromatography (HPLC). Although the role of the mobile phase in chromatographic retention and selectivity has been extensively studied, that of the stationary phase has only come under intense scrutiny recently. As a result, the effects of the stationary phase on chromatographic properties is not yet fully understood.
The stationary phases are generally prepared by converting, e.g., by hydrolysis, the oxide groups on the metallic oxide to hydroxyl groups and reacting the latter with reactive silanes, e.g., silyl halides to produce the silylated metal oxide and, in the case of a halide reactant, a hydrogen halide by-product.
One reason for this dearth of knowledge as to the effect of the stationary phases on chromatographic properties is the lack of precise and reliable methods for determining bonded phase characteristics such as the density, homogeneity and topographical distribution of the bonded silyl ligands and the residual hydroxyl groups on the support surface. These properties are a direct consequence of the bulk metallic oxide medium and the reagent and reaction conditions for the silanization process [Kinkel et al, J. Chromatogr., Vol. 316, pp 193-200 (1984)]. In order to obtain reversed phase packings with reproducible surface characteristics, the silanization reaction conditions must be painstakingly controlled. Careful optimization of these reaction conditions makes possible the reproducible synthesis of bonded phases.
In the preparation of reversed phase packings, one objective is the modification of as many surface hydroxyl groups on the silica as possible, especially the highly acidic isolated silanols. These residual isolated silanol groups have been shown to be the main cause of tailing of chromatographic peaks for basic compounds, of mechanical instability for the packing, and of low sample capacity for the column [Kohler et al, J. Chromatogr., Vol. 352, pp. 275-305 (1986); Kohler et al, J. Chromatogr., Vol. 385, pp. 125-150 (1987)]. Di- or tri-reactive alkylsilanes had previously found favor over monoreactive silanes because of their greater reactivity and the possibility of reacting simultaneously with two or three hydroxyl groups. However, any unreacted sites on the bonded functional groups will be hydrolyzed upon contact with water (i.e., from the mobile phase), forming additional undesirable silanol groups [Snyder et al, "Introduction to Liquid Chromatography", 2nd ed., Wiley-Interscience: N.Y., 1979; Chapter 7; Berendsen et al, J. Liq. Chromatogr., Vol. 1, pp. 561-586 (1980)]. Di- and tri-reactive silane reagents also often result in nonreproducible stationary phases since the degree of polymerization is highly dependent on the residual water content of the metal oxide and the reagents used in the bonding reaction [Snyder, supra]. Another drawback of polymeric stationary phases is their lower chromatographic efficiency, which results from poor solute mass transfer in these relatively thick stationary phases. Therefore, many investigators now advocate the use of monofunctional silanes for the derivatization reaction, since this results in a reproducible and well-defined chemically bonded phase. Additionally, monomeric stationary phases generally exhibit superior column performance to polymeric phases due to their faster solute mass transfer kinetics [Cooke et al, J. Chromatogr., Vol. 18, pp. 512-524 (1980)]. For the derivatization of silica with octadecyldimethylchlorosilane, the most commonly used monoreactive silane, the resulting bonding reaction may be depicted thusly: ##STR1## where M is a metal.
Kinkel and Unger, supra, have studied the roles of the solvent and the base in these monofunctional bonding reactions and have found their choice to be crucial. When alkylhalosilanes are reacted with silica, a base is added to serve as the acid-acceptor catalyst, binding the haloacid formed during the reaction and driving the equilibrium to the product side. In addition, the base favorably affects the kinetics of the silanization reaction. Mechanistic studies of these types of reactions [Corriu et al, J. Organomet. Chem., Vol. 198, pp. 321-320 (1980)] have shown that two molecules of base attack one molecule of silane, activating the Si-X bond such that a reactive intermediate and a hydrohalide are formed. Formation of this reactive intermediate greatly increases the kinetics of the bonding reaction; indeed, the addition of the acid-acceptor catalyst results in approximately 90% of the total conversion taking place within the first hour of the reaction [Kinkel et al, supra]. In their study, Kinkel and Unger found that the two most effective acid-acceptor catalysts for organohalosilanes were imidazole and 2,6-lutidine.
The reaction solvent must also be carefully chosen. The solvent can interact specifically with the silane, the base and the surface silanol groups on the silica. When the solvent interacts with a silanol group, there is a considerable effect on the strength of the bond between the silicon and oxygen atoms. Solvents which have both a pronounced Lewis acid and Lewis base character cause the SiO bond strength to be weakened and facilitate the bonding reaction. The solvent can also activate the silicon atom of the organohalosilane by forming a pentacoordinated intermediate through nucleophilic attack. The resultant bond lengthening causes nucleophilic activation to occur, favoring attack by a second nucleophile (such as the base). The solvent may influence the base as well, as it is known that in aprotic polar solvents the nucleophilic character of reactants is more pronounced. All of these considerations may have a synergistic relationship as well. Based on their experimental work with organohalosilanes, Kinkel and Unger found that methylene chloride and N,N-dimethylformamide were the most effective solvents for the bonding reaction.
Many organic reactions have been shown to be enhanced by ultrasound [Lorimer et al, Chem Soc. Rev , Vol. 16, pp. 239-274 (1987); Lindley et al, Chem. Soc. Rev., Vol. 16, pp. 275-311 (1987); Bremner, Chem Br., Vol. 22, pp. 633-638 (1986); Boudjouk, J. Chem. Educ., Vol. 63, pp. 427-429 (1986); Han et al, Organometallics, Vol. 2, pp. 769-771 (1983); Clough et al, J. Chem. Educ., Vol. 63, p. 176 (1986); Suslick, Mod. Synth. Methods, Vol. 4, pp. 1-60 (1986). Boudjouk and Han have shown [Boudjouk et al, Tetrahedron Lett., Vol. 22, pp. 3813-3814 (1981)] that in the presence of ultrasonic waves both alkyl and aryl chlorosilanes could be coupled over lithium wire; without ultrasonification, this reaction occurred to no appreciable extent. Reactions at solid-liquid interfaces have also been shown to be enhanced by ultrasound [Bremner, supra; Suslick, supra].
It is an object of the present invention to provide a novel reversed phase stationary phase for liquid chromatography and an improved method for the manufacture thereof.